Summary

The spinal muscular atrophy (SMA) gene product SMN forms with gem-associated protein 2–8 (Gemin2–8) and unrip (also known as STRAP) the ubiquitous survival motor neuron (SMN) complex, which is required for the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), their nuclear import and their localization to subnuclear domain Cajal bodies (CBs). The concentration of the SMN complex and snRNPs in CBs is reduced upon SMN deficiency in SMA cells. Subcellular localization of the SMN complex is regulated in a phosphorylation-dependent manner and the precise mechanisms remain poorly understood. Using co-immunoprecipitation in HeLa cell extracts and in vitro protein binding assays, we show here that the SMN complex and its component Gemin8 interact directly with protein phosphatase PP1γ. Overexpression of Gemin8 in cells increases the number of CBs and results in targeting of PP1γ to CBs. Moreover, depletion of PP1γ by RNA interference enhances the localization of the SMN complex and snRNPs to CBs. Consequently, the interaction between SMN and Gemin8 increases in cytoplasmic and nuclear extracts of PP1γ-depleted cells. Two-dimensional protein gel electrophoresis revealed that SMN is hyperphosphorylated in nuclear extracts of PP1γ-depleted cells and expression of PP1γ restores these isoforms. Notably, SMN deficiency in SMA leads to the aberrant subcellular localization of Gemin8 and PP1γ in the atrophic skeletal muscles, suggesting that the function of PP1γ is likely to be affected in disease. Our findings reveal a role of PP1γ in the formation of the SMN complex and the maintenance of CB integrity. Finally, we propose Gemin8 interaction with PP1γ as a target for therapeutic intervention in SMA.

Introduction

The mammalian cell nucleus is organized into distinct compartments (Mao et al., 2011) and the Cajal body (CB) is one of these entities (Morris, 2008). Most cells contain two to four CBs that are identified by the protein marker coilin (Raška et al., 1991). Coilin has been conserved in evolution, and various studies suggest that coilin and CBs are not essential in fruit fly and mice, whereas they are in developing zebrafish embryos (Strzelecka et al., 2010). CBs are prominent in cancer cells and neurons (Lafarga et al., 2009). They are highly dynamic subnuclear domains enriched in many different ribonucleoproteins (RNPs), such as spliceosomal small nuclear RNPs (snRNPs), small nucleolar RNPs (snoRNPs), RNA polymerases, histone mRNA processing factors and telomerase (Cioce and Lamond, 2005; Gall, 2000; Matera et al., 2007).

The biogenesis of snRNPs involves cytoplasmic and nuclear phases. Synthesis starts with the transcription of a small nuclear RNA (snRNA) precursor and CB targeting to associate with the nuclear export complex (Suzuki et al., 2010). In the cytoplasm, survival motor neuron (SMN) complex assembles the snRNAs with common Sm core proteins into core snRNPs. Following nuclear import, core snRNPs are targeted to CBs for additional maturation and processing steps resulting in the production of mature snRNPs for splicing in the nucleoplasm or for storage in speckles. CBs have also been reported to increase assembly and recycling of snRNPs after splicing (Nesic et al., 2004; Klingauf et al., 2006; Stanek et al., 2008). The SMN complex could also operate in these processes (Pellizzoni et al., 1998).

The severity of MN diseases correlates with a deficiency of SMN in the nucleus (Turner et al., 2009). The precise nuclear function of SMN is unknown. Nuclear import of the SMN complex depends on snRNPs (Narayanan et al., 2004) and targeting to CBs on its interaction with WD40 encoding RNA antisense to p53 (WRAP53; also known as telomerase Cajal body protein 1), a factor involved in CB recruitment of small CB-specific RNAs (Mahmoudi et al., 2010). SMN has also been reported to form a different type of nuclear body designated a ‘gem’ for Gemini of CBs (Liu and Dreyfuss, 1996). Gems contain the components of the SMN complex and lack coilin and snRNPs. Depletion of SMN disrupts CBs and gems, whereas coilin depletion disrupts CBs and forms gems (Lemm et al., 2006; Whittom et al., 2008). These data indicate that SMN is required for CB formation, and gems form when snRNP metabolism is disturbed. Thus, gems are thought to be storage sites. The tethering reaction of SMN has been reconstituted in cells, showing de novo CB formation as a non-linear assembly process triggered by most components when SMN is present at sufficient levels (Kaiser et al., 2008). However, the mechanisms controlling the accumulation in CBs are so far not fully understood.

Many forms of stress, stimuli or signaling pathways impact on CB composition, size and number (Boulon et al., 2010). These processes result in substantial changes in protein–protein interactions influenced by post-translational modifications, such as protein methylation (Boisvert et al., 2002; Hebert et al., 2002; Clelland et al., 2009) and phosphorylation (Hebert, 2010). SMN and coilin are phosphoproteins. The phosphorylation of SMN regulates the stability of the complex (Burnett et al., 2009) and snRNP assembly in the cytoplasm (Grimmler et al., 2004). Hyperphosphorylation of coilin coincides with lack of CBs during mitosis (Carmo-Fonseca et al., 1993) and SMN preferentially interacts with the hypophosphorylated coilin that forms CBs (Hebert et al., 2002). The nuclear protein phosphatase PPM1G, which is not present in CBs, has been reported as contributing to the dephosphorylation of SMN and coilin and to their accumulation in CBs (Hearst et al., 2009; Petri et al., 2007). Other studies on CBs have implicated phosphorylation: inhibiting phosphatases and a phosphoserine mutation in coilin affect the CB localization of coilin and of snRNPs (Lyon et al., 1997; Sleeman et al., 1998). The nuclear phosphatase PP4 associates with the SMN complex and enhances the temporal CB localization of snRNPs (Carnegie et al., 2003). Moreover, inhibition of PP1 has been shown to increase the number of gems in SMA fibroblasts (Novoyatleva et al., 2008). How PP1 could regulate SMN localization is unknown.

PP1 holoenzymes regulate a wide range of cellular functions, including transcription, pre-mRNA splicing, cell survival, synaptic plasticity and muscle contraction (Ceulemans and Bollen, 2004; Moorhead et al., 2007). In mammals, there are three PP1 catalytic subunits (α, β/δ and γ), each encoded by a different gene. The association with a regulatory (or targeting) subunit determines the subcellular localization and substrate specificity of the catalytic subunits (Cohen, 2002; Bollen and Beullens, 2002). The list of approximately 180 regulatory subunits is still growing (Bollen et al., 2010). Most targeting proteins harbor a primary consensus PP1-binding ‘R/KVxF’ motif (Meiselbach et al., 2006) and the localization pattern of each catalytic subunit results from the addition of many different PP1 holoenzyme complexes. All three localize to the cytoplasm and nucleus during interphase. PP1α and PP1γ are found in the nucleoplasm and PP1γ also concentrates in nucleoli, whereas PPP1β/δ is detected throughout the nucleus with no particular accumulation pattern (Andreassen et al., 1998). The PP1 catalytic subunits are highly mobile and their subcellular localization could change with the expression levels of the targeting proteins (Trinkle-Mulcahy et al., 2001; Trinkle-Mulcahy et al., 2003). One of the major nuclear targeting subunits of PP1α and PP1γ is the phosphatase nuclear targeting subunit PNUTS [also known as serine/threonine-protein phosphatase 1 regulatory subunit 10 (PP1R10) and p99] that localizes to the nucleoplasm (Landsverk et al., 2005) and can be found occasionally in CBs (Moorhead et al., 2007). These data illustrate how dynamic and transient the targeting of PP1 to different specific subcellular sites could be.

We report here a previously unknown mechanism of post-translational regulation of the SMN complex by PP1γ that was determined using biochemical, proteomic, RNA interference and immunofluorescence approaches. For in vivo immunofluorescence experiments involving control and SMA individuals we chose the skeletal muscle because of the very specific cytoplasmic organization. We examined the proteins co-immunopurified with SMN proteins and focused on a new interaction with PP1γ. We identified Gemin8, a component of the SMN complex, as a PP1-binding protein targeting PP1γ to CBs. We show that modulating the expression levels of PP1γ impact on the subnuclear organization. Our results support the conclusion that PP1γ could regulate SMN complex formation and localization to CBs.

Results

Proteomics study of the N-terminal region of SMN

We previously produced SMN deletion mutants in order to gain insight into its subnuclear localization in mammalian cells (Renvoisé et al., 2006). A diagram of the domains used in the present study for fluorescent fusion proteins is shown in Fig. 1A. The fragment containing the lysine-rich region [amino acids (aa) 71–83] and Tudor domain (aa 91–151) produced a fusion protein GFP–SMN472Δ5 (hereafter referred to as GFP–472Δ5) that localizes to the nucleoplasm and CBs. Introduction of mutations in the lysine-rich region of GFP–472Δ5 abolishes the localization in CBs. We expressed the N-terminal region of SMN as a GFP fusion protein (GFP–N86; 86 first aa) and performed immunoprecipitation experiments with anti-GFP antibody to identify new interaction partners of SMN (Fig. 1B,C). In co-immunoprecipitated proteins, we observed a major band of approximately 40 kDa corresponding to the apparent mass of GFP–N86 (Fig. 1C). The band was cut, digested and analysed by mass spectrometry (MS). Peptides from GFP–N86 and from protein phosphatase PP1γ, as a candidate partner of expected mass, were detected (supplementary material Fig. S1). We sought to determine the relationship between PP1γ and SMN because SMN complex localization and function are regulated by protein phosphorylation.

Interaction of SMN and PP1γ assayed by co-immunoprecipitation. (A) Schematic representation of SMN (aa 1–294) and mutants fused to eGFP at the N-terminus (Renvoisé et al., 2006). SMN is depicted with the Lys-rich, Tudor, Pro-rich, YG-box and exon7-encoded domains. (B) Flow-chart of the immunopurification procedure. (C) Immunoprecipitations (IPs) were performed with negative control mouse immunoglobulins (IP Control) or anti-GFP (IP-GFP) antibody and with extracts from COS cells expressing eGFP-tagged N86. Bound proteins were separated by SDS-PAGE and visualized with silver staining. An arrow indicates a 40-kDa band. The asterisk indicates the expected position of the SMN complex component Gemin8. (D) IPs were performed with extracts from COS cells stably expressing eGFP-tagged 472Δ5, N86 or double mutant N86M2, respectively. Bound proteins were analysed by immunoblotting with anti-PP1γ antibodies. The anti-GFP incubation served as a loading control. (E) Bound proteins after IP of GFP–SMN were analysed for PP1γ and endogenous SMN. The association of PP1γ with the fusion proteins requires the Lys-rich domain of SMN. IgGh and IgGl are the immunoglobulin heavy and light chains, respectively.

Co-immunoprecipitation of PP1γ with the SMN complex

To test the association of PP1γ with SMN, we expressed GFP–472Δ5, GFP–N86 and GFP–N86M2 fusion proteins and performed co-immunoprecipitation experiments (Fig. 1D). Immunoblot analyses revealed that PP1γ was co-immunoprecipitated with GFP–472Δ5 and GFP–N86. We found little if any interaction between PP1γ and GFP–N86M2, a fusion protein in which the lysine residues are mutated (Renvoisé et al., 2006). This mutation disrupts a minor self-interacting motif of SMN that could regulate stability and CB localization of the SMN complex (Morse et al., 2007). Interaction between endogenous SMN and PP1γ proteins appeared to be less efficient (data not shown) than between GFP–SMN and PP1γ (Fig. 1E), suggesting that either the anti-SMN antibody masks the interaction site or a third component mediates the interaction between them.

Direct interaction of PP1γ with SMN complex and its component Gemin8

A pull-down assay was used to demonstrate direct interaction between PP1γ and the SMN complex (Fig. 2). Anti-SMN antibody 4B3 allowed us to purify the SMN complex from HeLa cell lysates under stringent conditions in order to remove any interacting proteins of weak affinity for the complex (Fig. 2A). The antibody-immobilized complex (SMN complex) was incubated with a purified Escherichia coli recombinant histidine-tagged PP1γ (His–PP1γ). After extensive washes, the proteins bound to the complex were eluted and tested for the presence of His–PP1γ by immunoblotting (Fig. 2B). The bound proteins were compared with that bound to unrelated mouse monoclonal antibody (control). The recombinant PP1γ was bound to the SMN complex. We then sought to identify the component of the complex that mediates binding to PP1γ. The most common PP1-binding RVxF motif has the consensus sequence [R/K]x0–1[V/I]{P}[F/W] (x represents any amino acid, and {P} any except proline). Based on the hypothesis that SMN might be a substrate for PP1γ, the sequences of SMN and of gemins that bind directly to SMN, namely Gemin2, 3 and 8, were examined. The HVAW motif in Gemin8 (aa 80–83) appeared to be a potential PP1-binding motif. Direct interaction between Gemin8 and PP1γ was tested using purified bacterial recombinant proteins (Fig. 2C). The immobilized glutathione S-transferase (GST)-tagged Gemin8 retained His-PP1γ, whereas the GST alone did not (Fig. 2D). Direct interaction between Gemin8 and PP1γ was further tested in fixed HeLa cells using the in situ proximity ligation assay (in situ PLA; Fig. 2E). This approach detects endogenous protein–protein interactions in fixed cells using antibodies specific for each protein (Fredriksson et al., 2002). Using in situ PLA, the Gemin8–PP1γ interaction appeared as bright dots in the cytoplasm, nucleoplasm and CBs. Moreover, endogenous Gemin8 interacted with PP1γ and SMN in co-immunoprecipitation experiments (Fig. 2F). Our results support the conclusion that Gemin8 is a PP1-binding protein.

Direct interaction of PP1γ with the SMN complex and its component Gemin8. (A) HeLa cell lysate was immunoprecipitated with negative control (IP Control) or anti-SMN antibody (IP SMN). Proteins were eluted, resolved by SDS-PAGE and analysed by silver staining. (B) Immunoprecipitated proteins shown in A were incubated with purified E. coli recombinant His-tagged PP1γ. Bound proteins were analysed by immunoblotting. (C) GST and GST–Gemin8 fusion proteins were purified, separated by SDS-PAGE and analysed by Coomassie staining. (D) GST fusion proteins shown in C were incubated with purified His-tagged PP1γ. Bound proteins were analysed by immunoblotting. PP1γ interacts directly with Gemin8. (E) In situ PLA detection of the interaction between endogenous proteins in HeLa cells with anti-Gemin8 and anti-PP1γ antibodies. As a control, one of the primary antibodies was omitted. Scale bar: 3 µm. (F) Co-IP of the endogenous proteins with anti-Gemin8 antibody and HeLa cell extracts were analysed by immunoblotting with anti-PP1γ and anti-SMN antibodies. The asterisks indicate the immunoglobulins.

Overexpression of Gemin8 colocalizes PP1γ with CB markers

Given that PP1-binding proteins confer subcellular localization and substrate specificity to PP1 holoenzymes, Gemin8 was expressed as a fluorescent fusion (FP–Gemin8; Fig. 3A) and its colocalization with SMN and PP1γ was tested. FP–Gemin8 completely colocalized with SMN to the cytoplasm and nuclear CBs (Fig. 3B), as reported (Carissimi et al., 2006). In FP–Gemin8-expressing cells, PP1γ was concentrated in numerous nuclear bodies with the fusion, whereas it was concentrated in nucleoli of untransfected cells and cells expressing either FP–SMN, myc–Gemin4 or FP alone (Fig. 3C). Colocalization with FP–Gemin8 could be due to the presence of other PP1-binding proteins in CBs. The C-terminal region of Gemin8 including the coiled coil domain (FP–G8ΔC) was deleted to reduce nuclear body formation and we introduced mutations in the putative PP1-binding motif (FP–G8ΔC3A; Fig. 3A,C). Both FP–G8ΔC and FP–G8ΔC3A localized to the nucleoplasm and to fewer CBs. FP–G8ΔC recruited PP1γ into CBs, whereas FP–G8ΔC3A did not. These experiments revealed that the HVAW motif of Gemin8 is required for the localization of PP1γ to CBs. Altogether, our results support the conclusion that Gemin8 contributes to CB localization of PP1γ in mammalian cells.

Targeting of PP1γ to Cajal bodies in cells overexpressing Gemin8. (A) Schematic representation of Gemin8 (aa 1–242) and mutants fused to destabilized YFP (FP) at the N-terminus. Gemin8 is depicted with the putative PP1-binding motif HVAW and a self-association coiled-coil domain (accession number Q9NWZ8). The expressed fusion proteins were assayed in total cell lysates by immunoblotting with anti-FP antibody. The asterisk indicates an unspecific protein band from HeLa cells. (B) Immunofluorescence experiments were performed in HeLa cells expressing FP–Gemin8 fusion protein with anti-SMN antibody. FP–Gemin8 colocalizes with SMN in the cytoplasm and nuclear bodies as shown in yellow (merged images) in low- and high-expressing cells. (C) FP–Gemin8, FP–G8ΔC, FP–G8ΔC3A, FP–SMN, myc–Gemin4 and FP alone were expressed and analysed by immunofluorescence with anti-PP1γ antibody. Overexpression of FP–Gemin8 changes the localization of PP1γ from nucleoli to Gemin8-expressing nuclear bodies in a manner dependent on the HVAW motif of Gemin8. Arrowheads point to untransfected cells and arrows to cells expressing fusions. Scale bar: 3 µm.

To determine whether the FP–Gemin8 nuclear bodies contained other CB constituents, colocalization with coilin (a CB marker), Gemin3 and components of snRNPs was examined (Fig. 4A–E). All FP–Gemin8 nuclear bodies were CBs as indicated by complete colocalization with coilin. They were also enriched in Gemin3 and snRNP-specific markers, such as the trimethylguanosine (TMG)-capped snRNAs (molecular signature for the nuclear import of snRNAs), snRNP Sm core protein SmD3 (one of the seven Sm proteins common to all snRNPs except U6 snRNP) and U1-specific 70 kDa protein. The localization of the abundant splicing factor SC35 that is a marker of speckles (Spector, 1993) and does not concentrate in CBs was investigated. Fig. 4F showed that SC35 staining occurs in speckles and not in CBs of FP–Gemin8-expressing cells, supporting the conclusion that the accumulation of PP1γ in CBs is specific. Our results demonstrated that Gemin8 could form CBs, which contain SMN complex, snRNPs and PP1γ.

Depletion of PP1γ enhances the accumulation of SMN complex and snRNPs in CBs

To determine whether there might be a functional link between PP1γ and SMN targeting to CBs, RNA interference (RNAi) knockdown experiments were performed using three distinct PP1γ-specific small interfering RNA (siRNA) duplexes (Fig. 5). We first tested the protein levels of total lysates of HeLa cells transfected with either a negative control firefly luciferase (Gl2) or PP1γ siRNAs (si#5, si#6 and si#8) by immunoblotting. The PP1γ-specific siRNAs reduced PP1γ protein levels by ∼65% (Fig. 5A). SMN complex levels remained the same, indicating the specificity of the siRNAs used to reduce PP1γ levels. As a control, the overall phosphorylation profile of total protein lysates was examined (Fig. 5A, furthest right panel). There were no major differences between PP1γ-depleted cell lysates and controls, indicating that PP1 holoenzymes have overlapping substrates. The localization of SMN in siRNA-treated cells was then examined by immunofluorescence experiments (Fig. 5B). Depletion of PP1γ (as shown by the reduced PP1γ staining) led to a significant (P<0.001) increase in the proportion of cells with more SMN nuclear bodies than control cells (Fig. 5B,C). There was a three- to fourfold increase in the proportion of cells with more than five nuclear bodies. All three PP1γ siRNAs confirmed the formation of CBs upon depletion of PP1γ (Fig. 5C). Colocalization experiments with coilin and CB components, including Gemin3, Gemin8 and snRNP-specific markers, all had similar results (Fig. 6A–F). These data indicate that depletion of PP1γ increases CB numbers and has no adverse effect on their composition.

Depletion of PP1γ enhances the accumulation of SMN nuclear bodies. HeLa cells were transfected with negative control Gl2 (GL2; firefly luciferase) or three different PP1γ siRNAs (#5, #6 and #8). (A) Cell lysates were analysed by immunoblotting using antibodies against components of the SMN complex. Incubation of α-tubulin served as a loading control. siRNA #5, #6 and #8 reduced the levels of PP1γ by 69, 64 and 64%, respectively (n = 5 experiments). The immunoblot in the right panel shows that lowering PP1γ levels (siRNA#5) had no major effect on the overall phosphoserine profile. (B) Double-labeling immunofluorescence experiments were performed using anti-PP1γ (red) and anti-SMN antibodies (green). The arrows point to silenced cells. The asterisk indicates an unsilenced cell. The microscope was focused on the nuclear foci. Scale bar: 3 µm. (C) Statistical analyses of SMN nuclear bodies in silenced cells (n>1000 cells). Error bars indicate the standard error of the mean (s.e.m.); ***P<0.001 (Student's t-test).

In the nucleus, SMN is localized with coilin in CBs and in gems, which lack coilin and snRNPs (Liu and Dreyfuss, 1996). To examine whether SMN localizes to either type of nuclear body in PP1γ-depleted cells, triple immunolabelling experiments were performed with PP1γ, SMN and coilin antibodies (Fig. 7A–D). In control Gl2 siRNA-treated cells SMN localized to both gems and CBs (Fig. 7A), whereas depletion of PP1γ led to complete localization of SMN to CBs (Fig. 7B). To demonstrate the specificity of the effects observed above, FP–PP1γ was expressed using a cDNA resistant to siRNA#5 (Fig. 7C). FP–PP1γ expression restored the localization of SMN to gems and CBs, whereas FP alone did not (Fig. 7D). Statistical analyses confirmed these results (Fig. 7E). The increased number of CBs observed in PP1γ-depleted cells was significantly (P<0.001) reduced in rescue experiments, indicating that the siRNA-mediated effects are specific of PP1γ. Our results support the conclusion that localization of SMN complex in either CBs or gems involves PP1γ.

Depletion of PP1γ enhances CB accumulation and the post-translational modification status of SMN. Triple-labelling immunofluorescence experiments were performed with anti-PP1γ, anti-SMN and anti-coilin antibodies on HeLa cells transfected with (A) negative control Gl2 (GL2) or (B) PP1γ siRNA#5. (C) The PP1γ-silenced cells were transfected with FP–PP1γ or (D) FP alone. The arrowheads and arrows point to one of the nuclear body gem (SMN alone) and to one of the CBs (SMN and coilin), respectively. The microscope was focused on the nuclear foci. Scale bar: 3 µm. (E) Statistical analyses of SMN nuclear bodies in silenced cells. Error bars indicate the s.e.m.; ***P<0.001 (Student's t-test). (F) HeLa cells were transfected with negative control (control RNAi) or PP1γ siRNA (PP1γ RNAi). Cell lysates were analysed by immunoblotting with PP1γ, PP1α and SMN antibodies. The anti-α-tubulin incubation served as a loading control. (G) Nuclei preparations from cells analysed in F were separated by conventional 2D gel electrophoresis (IF; followed by second dimension, SDS-PAGE). Immunoblots were revealed with anti-SMN antibody. Brackets indicate four sections with major changes upon PP1γ knockdown and rescue. The highest to the lowest phosphorylated forms of SMN are from section 1 to 4. The arrowheads point to isoforms accumulated in PP1γ-depleted nuclei that disappeared when FP–PP1γ was expressed. The arrows point to isoforms absent in PP1γ-depleted nuclei and restored in rescue experiments. (H) The relative intensities of signals detected in sections of the SMN immunoblots shown in G.

Depletion of PP1γ changes the post-translational modification pattern of SMN

To attempt to understand how PP1γ is involved in CB localization of the SMN complex as demonstrated above, we sought to determine whether depletion of PP1γ might influence the phosphorylation status of SMN. To test the hyperphosphorylation hypothesis, SMN immunoblotting of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) gels was performed. As a control, protein levels in siRNA-transfected cell lysates were examined (Fig. 7F). The PP1γ siRNA led to PP1γ reduction when compared with Gl2 siRNA (control RNAi). Transfection of PP1γ siRNA#5 alone or with a FP–PP1γ expression plasmid resistant to the siRNA#5 did not reduce either PP1α (PP1 catalytic subunit alpha) or SMN levels, again indicating the specificity of this approach. The post-translational modification patterns of SMN from nuclear extracts of transfected cells were compared and relative signal intensities were measured (Fig. 7G,H). A complex pattern of numerous SMN isoforms was detected in control conditions as reported previously (Grimmler et al., 2004). Examination of SMN isoforms from PP1γ-depleted cell extracts revealed striking differences. First, SMN phosphorylation increased upon depletion of PP1γ as indicated by the accumulation of more acidic isoforms (section 1), whereas expression of FP–PP1γ reduced them. Second, the spots at pH 6.8 (section 2) and pH 8.5 (section 3) that were reduced upon PP1γ depletion (Fig. 7G, arrows) were also rescued by FP–PP1γ. Third, a basic isoform at pH 9.2 (section 4, arrowhead) accumulated in PP1γ-depleted extracts, whereas FP–PP1γ caused it to disappear, and a slightly less basic spot to form. Finally, overall populations of spots from section 2–4 were partially rescued by FP–PP1γ. Our results support the conclusion that PP1γ regulates changes in the post-translational modification pattern of SMN.

Depletion of PP1γ results in an increased association of SMN and Gemins

We tested the possibility that the increased number of CBs demonstrated above in PP1γ-depleted cells might correlate with accumulation of the SMN complex in the nucleus. Cytosolic and nuclear fractions were prepared from HeLa cells transfected with either Gl2 or PP1γ siRNAs and the distribution of the SMN complex was monitored by immunoblotting (Fig. 8A). It was not possible to probe for Gemin5, -6 and -7 because of the limited affinities of commercial antibodies. Gemin8 and unrip were predominantly cytosolic as reported (Carissimi et al., 2005; Carissimi et al., 2006; Grimmler et al., 2005). There was no major change in the nucleocytoplasmic distribution of the SMN complex, indicating that PP1γ is not involved in this process.

Depletion of PP1γ promotes formation of the SMN complex. (A) HeLa cells were transfected with control Gl2 or three different PP1γ siRNAs (#5, #6 and #8) and separated into cytosolic and nuclear fractions. The fractions were analysed by immunoblotting with antibodies against components of the SMN complex. The anti-α-tubulin and anti-HDAC incubations served as loading controls for the cytosolic and nuclear fractions, respectively. (B) The cytosolic fractions of HeLa cells transfected with Gl2 or PP1γ siRNA #5 were immunoprecipitated with control mouse immunoglobulins (Ctrl) and anti-SMN antibody (SMN). The immunoprecipitates were analysed by immunoblotting. (C) The relative amounts of the proteins associated with SMN were estimated using the relative band intensities detected by immunoblotting, normalized to the SMN band. The histograms show the results of five experiments. Error bars indicate the s.e.m. *P<0.042 (Wilcoxon signed-rank test). (D) Similar analyses of the SMN complex in the nucleus.

Given the role of phosphorylation in protein–protein interactions, we tested whether PP1γ might regulate the association of the Gemins with SMN. To this effect, cytosolic and nuclear fractions were immunoprecipitated using anti-SMN antibodies, and co-immunoprecipitated proteins were analysed by immunoblotting (Fig. 8B–D). In SMN complexes from the cytosol of PP1γ-depleted cells, Gemin4 and -8 and unrip were significantly (P<0.042) increased relative to SMN, whereas Gemin2 and -3 were not (Fig. 8C). Similar changes were found when the nuclear SMN complexes were examined (Fig. 8D). This indicated no major change in the association of SMN–Gemin2–Gemin3 subcomplex, whereas the association of the peripheral Gemins changed. As described previously (Carissimi et al., 2006; Charroux et al., 2000; Otter et al., 2007), SMN interacts directly with Gemin8, and Gemin3 with Gemin4. Our results support the conclusion that PP1γ regulates these interactions.

PP1γ and Gemin8 colocalize in the cytoplasm of skeletal muscles

As a first approach to understand the in vivo physiological relevance of the interaction between Gemin8 and PP1γ demonstrated here, we sought to determine their colocalization in mammalian tissue sections. The skeletal muscle was chosen for its specific subcellular organization and localization of the SMN complex at the Z-discs of mouse myofibrils (Walker et al., 2008). Using the Prestige anti-Gemin8 antibodies validated by the Human Protein Atlas project in numerous tissues including the skeletal muscle (www.proteinatlas.org), the expected striated pattern of Z-discs was found on 14-day post-natal mouse and human foetal skeletal muscles (Fig. 9A,B). Our colocalization experiments revealed substantial, although not complete, overlap of PP1γ with Gemin8. These results support the conclusion that Gemin8 and PP1γ could colocalize in vivo.

Localization of Gemin8 and PP1γ in skeletal muscles. (A,B) Subcellular localization of Gemin8 and PP1γ in longitudinal mouse and human foetal skeletal muscle sections as shown by immunofluorescence. Gemin8 has the striated appearance of the Z-discs as previously described for the components of the SMN complex (Walker et al., 2008). PP1γ colocalizes with Gemin8 at the Z-discs as shown in merged images (yellow). ‘2nd only’ are control images of muscle sections incubated with the two fluorescent secondary antibodies alone. (C) SMN deficiency altered the muscle structure organization and localization of Gemin8 and PP1γ in human Type I SMA foetuses. (D) Immunoblot analyses of muscle lysates from human control and type I SMA foetuses with Gemin8, SMN and PP1γ. The anti-α-tubulin incubation served as loading control. (E) Immunoprecipitation of Gemin8 from control and type I SMA patient fibroblasts were analysed by immunoblotting. (F) Statistical analyses of the relative levels of SMN and PP1γ co-precipitated with anti-Gemin8 antibody, and normalized to the levels of immunoprecipitated Gemin8 and to the levels of SMN input in samples (n = 3). Error bars indicate s.e.m.; **P<0.01 (Student's t-test).

Our previous studies showed a marked reduction of SMN levels in skeletal muscles from foetuses with severe SMA (Burlet et al., 1998) (Fig. 9D). Morphological defects consistent with a Z-disc deficiency have been reported in a mouse model with severe SMA (Walker et al., 2008; Kariya et al., 2008). To address the question of whether similar defects occur in the human disease, colocalization experiments were carried out on muscle sections from SMA foetuses (Fig. 9C). An altered organization of the striated pattern was observed as indicated by aberrant Gemin8 and PP1γ localization and a severe atrophy, reflecting denervated fibers. To provide molecular insights into pathobiology, control and severe SMA fibroblast cell extracts were immunoprecipitated using anti-Gemin8 antibodies and co-immunoprecipitated proteins were analysed by immunoblotting (Fig. 9E). As reported previously (Lefebvre et al., 2002), a reduction of SMN levels of ∼75% was detected in SMA fibroblast cell cultures compared with control fibroblasts. There was a significant (P<0.01) 60% reduction in interaction between SMN and Gemin8 or PP1γ in SMA fibroblasts, whereas no significant difference of the Gemin8 interaction with PP1γ was detected (Fig. 9F). This is consistent with the 50–70% reduction of in vitro snRNP assembly activity previously reported for SMA fibroblast cell extracts (Gabanella et al., 2007; Wan et al., 2005). Our results support the conclusion that the production of SMN complex is less efficient in SMA conditions.

Discussion

The SMN complex plays a central role in snRNPs biogenesis (Fischer et al., 1997) and in SMA, the second most frequent autosomal recessive disease of children (Lefebvre et al., 1995). snRNP biogenesis is fundamental for splicing and transcriptome integrity (Kaida et al., 2010). One key consequence of splicing activity in cells is the accumulation of SMN and snRNPs in CBs (Liu and Dreyfuss, 1996; Carvahlo et al., 1999), which is disrupted in SMA motor neurones (Lefebvre et al., 1997). Understanding the mechanisms underlying SMN interaction is important for cell survival in general and particularly for the development of SMA therapy. Using co-immunoprecipitation experiments we identified a previously unknown regulator of the SMN complex. Protein phosphatase PP1γ interacts with the complex and regulates its formation and localization to CBs.

Gemin8 binds directly to PP1γ and brings it into the multiprotein SMN complex, which is essential for snRNP biogenesis. SMN complex subunits associate in a stepwise manner in the cytoplasm at various stages of the snRNP biogenesis pathway (Carissimi et al., 2006; Massenet et al., 2002; Yong et al., 2010). Direct Gemin8 binding to SMN is at the heart of the association of SMN–Gemin2 and Gemin6–Gemin7–Gemin8–unrip subunits to form an intermediate complex that is competent to associate the Sm proteins of snRNPs. Thereafter, Gemin5, bound to the pre-snRNA, and Gemin3 and -4 are considered to separately join the intermediate complex. We observed here that the reduction of PP1γ levels enhances SMN interaction with Gemin8 and the number of CBs. As suggested by a mathematical model predicting that CBs accelerate snRNP biogenesis (Klingauf et al., 2006), our data indicate that PP1γ depletion enhances the cytoplasmic assembly of snRNPs and thus, their nuclear import and accumulation in CBs.

Our observations suggest that the formation of an intermediate complex made of SMN–Gemin2 and Gemin6–Gemin7–Gemin8–unrip is regulated by phosphorylation. We propose that PP1γ regulates the phosphorylation state of SMN to prevent premature formation of the intermediate complex. Although the exact PP1γ target sites of SMN are unknown, 2D-gel analyses reveal that lowering PP1γ levels lead to accumulation of hyperphosphorylated SMN (Fig. 7). Moreover, a reduction in the extent of SMN phosphorylation was observed by expressing PP1γ in PP1γ-depleted cells, which correlates with a lower CB number, reflecting downregulation of snRNP biogenesis. This provides a link between previous studies showing that SMN complex formation depends on SMN phosphorylation (Grimmler et al., 2004; Burnett et al., 2009) and its interaction with Gemin8 (Carissimi et al., 2006).

Other studies involving dephosphorylation of SMN in the nucleoplasm, showed that PP1MG regulates SMN complex stability in CBs (Petri et al., 2007). Consistent with our findings, PP1MG activity is probably not altered in PP1γ-depleted cells. The Gemin8 interaction with PP1γ in CBs (Fig. 2E) suggests that the two phosphatases function in different subnuclear compartments. Although SMN is the best candidate for a PP1γ substrate, other proteins interacting with Gemin8 or located in the vicinity could also be candidates. There are sixteen potential phosphoserines in SMN. It will be interesting to determine whether PP1γ targets the same residues as PP1MG and if not, this could explain why depletion of PP1MG decreases SMN localization to CBs (Petri et al., 2007), whereas PP1γ depletion increases it.

Regulation of SMN interactions could stabilize the complex (Burnett et al., 2009; Ogawa et al., 2009). SMA mutations in the C-terminal region prevent self-oligomerization (Lorson et al., 1998) that disrupts Gemin8 binding to SMN and hinder SMN complex formation (Otter et al., 2007). There is a second minor self-oligomerization domain lying in the Lys-rich domain of SMN (Morse et al., 2007). Indeed, PP1γ interaction with SMN complex appears to require two elements, the self-oligomerization Lys-rich domain of SMN (Fig. 1) and Gemin8 (Fig. 2). Dependence on the production of a functional SMN complex would provide a rationale for the failure of PP1γ to associate with a mutant lacking the self-oligomerization domains. Another possibility is that the phosphoserine S80-P (MS information, PhosphositePlus; http://www.phosphosite.org/) located within the Lys-rich domain might be sensitive to PP1γ. This aspect awaits further investigation.

It is worth noting that SMA modelling in Drosophila melanogaster and Caenorhabditis elegans has identified PP1 regulatory (inhibitory) subunit 13B (PP1R13B) as a modifier of SMN-deficiency phenotypes (Chang et al., 2008; Dimitriadi et al., 2010). This is consistent with our model in which ablation of the PP1 regulatory subunit exacerbates SMN-related defects by upregulating PP1. Only SMN and Gemins2, -3 and -5 has been identified in Drosophila (Kroiss et al., 2009). It is therefore conceivable that another SMN-associated protein binds PP1γ. In the human SMN complex, Gemin5 has a putative PP1-binding RVxF motif, but Gemin5 does not form CBs (Hao et al., 2007). Other PP1-binding motifs are Fxx[R/K]x[R/K] (Garcia et al., 2004) and DxxDxxxD (Neduva et al., 2005). The former motif occurs in Gemin4 and the latter in SMN. We have shown here that neither Gemin4 nor SMN recruits PP1γ to CBs (Fig. 3). Collectively, our results are most consistent with Gemin8 targeting PP1γ to CBs. The similarity of effects upon Gemin8 overexpression (Fig. 4) and PP1γ depletion (Fig. 6) indicate that Gemin8 is a previously unknown regulatory subunit of PP1.

Loss of SMN leads to motor neuron degeneration and muscle atrophy in SMA. The importance of SMN in muscles is beginning to be appreciated, given the varing degrees of disease severity (Bosch-Marcé et al., 2011; Kariya et al., 2008; Mutsaers et al., 2011). Although the mechanisms are still elusive, several observations can be considered. Lowering SMN in cultured cells alters fusion and morphology of differentiated myoblasts (Shafey et al., 2005). Ablation of the muscular SMN causes loss of regeneration by satellite cells (Nicole et al., 2003). Moreover, localization of the SMN complex with α-actinin at the level of Z-discs suggests a function outside snRNP biogenesis (Walker et al., 2008). Our observations that PP1γ and Gemin8 colocalize at Z-discs indicate that PP1γ could regulate a function of the SMN complex in vivo (Fig. 9). Lack of subcellular organization and aberrant localization of Gemin8 and PP1γ, as observed in SMA muscles, compares well with the Z-disc phenotype in SMA mouse models (Walker et al., 2008). Another interesting observation is that the interaction of PP1γ with SMN is reduced in SMA cells. These defects might impair the regulation of SMN complex in SMA.

A plausible strategy for the development of therapeutics for SMA is to increase SMN protein levels (Lorson et al., 2010). The physiological relevance of our data is that a specific manipulation of PP1γ might impact on the SMN complex independently of an increase in SMN levels (Figs 5, 8). However, pharmacological ablation of all three PP1 catalytic subunits causes formation of SMN nuclear gems in SMA cells (Novoyatleva et al., 2008; our unpublished data). Because gems are storage sites formed when pre-mRNA splicing is inhibited, more specific molecules will be required. Modeling the interaction shown here between PP1γ and the SMN complex could be a promising target in SMA therapy. Future in vivo experiments will need to address whether this has any beneficial effects.

Remarkable pleiotropy is seen for PP1 holoenzymes in distinct cellular pathways, including the EGF, TGF-β and FGF signaling pathways that have been linked to SMN (Bruns et al., 2009; Chang et al., 2008; Gangwani et al., 2001; Sen et al., 2011). Our studies reveal a regulation of the SMN complex and of nuclear body formation by PP1γ, identify Gemin8 as directly interacting with PP1γ, show mislocalization of PP1γ and Gemin8 in SMA muscles in vivo and altered interaction of PP1γ with SMN in SMA cells. A tight regulation of SMN complex assembly is revealed. It is therefore possible that imbalance of SMN sub-complexes produced by the reduction of SMN contributes to SMA severity.

Materials and Methods

Cell culture and transfections

COS7 and HeLa cell cultures were maintained in DMEM supplemented with 10% FBS, penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified CO2 incubator at 37°C. HeLa cells were plated in 60 mm dishes or eight-chamber culture slides (Becton Dickinson) for transfection using HiPerFect transfection reagent (Quiagen) with PP1γ-validated siRNAs (Hs_PPP1CC_5, _6 and _8) and negative controls Gl2 or scrambled duplexes (25 nM). For the rescue experiments, HeLa cells were first transfected with siRNA (Hs_PPP1CC_5) and 16 hours later the cells were transfected with pdEYFP-PP1γ plasmid (IOH14587-pdEYFPC1amp, imagines, Source BioScience, Nottingham, UK) using Transmessenger reagent (Qiagen). After a 5-hour incubation, the medium was replaced with fresh medium and incubated for a further 21 hours. Using FuGENE (Roche), HeLa cells were transfected with expression plasmids for pEGFP-SMN (Renvoisé et al., 2006), myc-Gemin4 (a gift from S. Massenet, CNRS, Nancy) and pdEYFP-Gemin8 (IOH3877-pdEYFP-C1amp; imaGenes), and pdEYFP-Gemin8ΔC or pdEYFP-Gemin8ΔC3A.

In situ proximity ligation assay

In situ proximity ligation assay (PLA) was performed as recommended by the manufacturer (DuolinkII kit, Olink Bioscience AB). Briefly, HeLa cells were fixed and permeabilized as above. Primary antibodies were diluted at 1.75 ng/µl rabbit anti-Gemin8 (Atlas antibodies, Sigma) and 2 ng/µl goat anti-PP1γ (C19, SC) in 1× antibody diluent and incubated for 30 minutes at room temperature. The negative control consisted of using only one primary antibody. The cells were washed twice for 5 minutes in Tris-buffered saline with 0.05% Tween. The PLA probes (Rabbit-MINUS and Goat-PLUS; Olink BioScience AB) were incubated for 90 minutes at 37°C. Subsequent steps were performed using the detection reagent Orange according to the DuolinkII kit protocol. In situ PLA signals were visible as dots with the RITC filter on the microscope. Nuclei were counterstained with DAPI to be able to select image position.

Statistical analyses of ECL signals were performed with ImageJ using grayscale images generated with an Epson Perfection 4990 PHOTO transparency scanner (1000 d.p.i. images, as shown in the figures assembled with Photoshop) or with a luminescent analyser LAS-3000 camera system (Fujifilm).

Acknowledgments

We wish to thank A. and J. Cartaud for constant support. We are grateful to colleagues for critical reading of the manuscript and generously providing protocols, controls and antibodies. S. Massenet (CNRS, Nancy) generously provided the myc-tagged Gemin4 plasmid. We acknowledge the proteomics core facility of the Institut Jacques Monod.

This work was supported by Institut National de la Santé et de la Recherche Médicale (salary to S.L.); Centre national de la recherche scientifique; the Association Française contre les Myopathies (grant to S.L.); an undergraduate fellowship from the Ministère de l'Education Nationale, de la Recherche et de la Technologie (to B.R.); and the Association Francaise Contre les Myopathies (to B.R.).

(1993). Assembly of snRNP-containing coiled bodies is regulated in interphase and mitosis--evidence that the coiled body is a kinetic nuclear structure.J. Cell Biol.120, 841–852.doi:10.1083/jcb.120.4.841